Mucopolysaccharidosis VI (MPS VI) is a rare lysosomal disease arising from impaired function of the enzyme Arylsulfatase B (ARSB). This causes aberrant accumulation of dermatan sulfate, a glycosaminoglycan (GAG) abundant in cartilage. While clinical severity varies along with age at first symptom manifestation, MPS VI usually presents early and strongly affects the skeleton. Current enzyme replacement therapy (ERT) does not provide effective treatment for the skeletal manifestations of MPS VI. This lack of efficacy may be due to inability of ERT to reach affected cells, or irreversibility of disease. To address the question of reversibility of skeletal phenotypes, we generated a conditional by inversion (COIN) mouse model of MPS VI, ArsbCOIN/COIN, wherein Arsb is initially null and can be restored to wild type using Cre. We restored Arsb at different times during postnatal development, using a tamoxifen-dependent global Cre driver. By restoring Arsb at postnatal days 7, 21, and 56-70 (P7, P21, and P56-P70), we determined that skeletal phenotypes can be fully rescued if Arsb restoration occurs at P7, while only achieving partial rescue at P21, and no significant rescue at P56-70. This work has highlighted the importance of early intervention in MPS VI patients to maximize therapeutic impact.
Elizabeth Hwang-Wong, Gabrielle Amar, Nanditha Das, Xiaoli Zhang, Nina A. Aaron, Kirsten Gale, Nyanza J. Rothman, Massimo Fante, Andrew D. Baik, Ajay Bhargava, Arun D. Fricker, Michelle McAlister, Jeremy S. Rabinowitz, John Lees-Shepard, Kalyan Nannuru, Aris N. Economides, Katherine D. Cygnar
Osteoarthritis (OA) is the most common joint disorder, and disease-modifying OA drugs (DMOADs) represent a major need in OA management. Krüppel-like factor 4 (KLF4) is a central transcription factor upregulating regenerative and protective functions in joint tissues. This study was aimed to identify small molecules activating KLF4 expression and to determine functions and mechanisms of the hit compounds. High-throughput screening (HTS) with 11,948 clinical-stage compounds was performed using a reporter cell line detecting endogenous KLF4 activation. Eighteen compounds were identified through the HTS and confirmed in a secondary screen. After testing in SW1353 chondrosarcoma cells and human chondrocytes, mocetinostat — a class I selective histone deacetylase (HDAC) inhibitor — had the best profile of biological activities. Mocetinostat upregulated cartilage signature genes in human chondrocytes, meniscal cells, and BM-derived mesenchymal stem cells, and it downregulated hypertrophic, inflammatory, and catabolic genes in those cells and synoviocytes. I.p. administration of mocetinostat into mice reduced severity of OA-associated changes and improved pain behaviors. Global gene expression and proteomics analyses revealed that regenerative and protective effects of mocetinostat were dependent on peroxisome proliferator-activated receptor γ coactivator 1-α. These findings show therapeutic and protective activities of mocetinostat against OA, qualifying it as a candidate to be used as a DMOAD.
Manabu Kawata, Daniel B. McClatchy, Jolene K. Diedrich, Merissa Olmer, Kristen A. Johnson, John R. Yates, Martin K. Lotz
Denosumab is an anti-RANKL antibody that potently suppresses bone resorption, increases bone mass, and reduces fracture risk. Discontinuation of denosumab causes rapid rebound bone resorption and bone loss but the molecular mechanisms are unclear. We generated humanized RANKL mice and treated them with denosumab to examine the cellular and molecular conditions associated with rebound resorption. Denosumab potently suppressed both osteoclast and osteoblast number in cancellous bone in humanized RANKL mice. The decrease in osteoclast number was not associated with changes in osteoclast progenitors in bone marrow. Long-term but not short-term denosumab administration reduced OPG mRNA in bone. Localization of OPG expression revealed that OPG mRNA is produced by a subpopulation of osteocytes. Long-term denosumab administration reduced osteocyte OPG mRNA suggesting that OPG expression declines as osteocytes age. Consistent with this, osteocyte expression of OPG was more prevalent near the surface of cortical bone in humans and mice. These results suggest that new osteocytes are an important source of OPG in remodeling bone and that suppression of remodeling reduces OPG abundance by reducing new osteocyte formation. The lack of new osteocytes and the OPG they produce may contribute to rebound resorption after denosumab discontinuation.
Qiang Fu, Nancy C. Bustamante-Gomez, Humberto Reyes-Pardo, Igor Gubrij, Diana Escalona-Vargas, Jeff D. Thostenson, Michela Palmieri, Joseph J. Goellner, Intawat Nookaew, C. Lowry Barnes, Jeffrey B. Stambough, Elena Ambrogini, Charles A. O'Brien
Reactive oxygen species (ROS) are natural products of mitochondrial oxidative metabolism and oxidative protein folding. ROS levels must be well controlled as elevated ROS has been shown to have deleterious effects on osteoblasts. Moreover, excessive ROS is thought to underly many of the skeletal phenotypes associated with aging and sex steroid deficiency in mice and humans. The mechanisms by which osteoblasts regulate ROS and how ROS inhibits osteoblasts are not well understood. Here, we demonstrate that de novo glutathione (GSH) biosynthesis is essential to neutralize ROS and establish a pro-osteogenic REDOX environment. Using a multifaceted approach, we demonstrate that reducing GSH biosynthesis leads to acute degradation of RUNX2, impaired osteoblast differentiation and reduced bone formation. Conversely, reducing ROS using Catalase enhances RUNX2 stability and promotes osteoblast differentiation and bone formation when GSH biosynthesis is limited. Highlighting the therapeutic implications of these findings, in utero antioxidant therapy stabilizes RUNX2 and improves bone development in the Runx2+/- haploinsufficient mouse model of human Cleidocranial Dysplasia. Thus, our data establish RUNX2 as a molecular sensor of the osteoblast REDOX environment and mechanistically clarifies how ROS negatively impacts osteoblast differentiation and bone formation.
Guoli Hu, Yilin Yu, Deepika Sharma, Shondra M. Pruett-Miller, Yinshi Ren, Guo-Fang Zhang, Courtney M. Karner
Hypochondroplasia (HCH) is a mild dwarfism caused by missense mutations in fibroblast growth factor receptor 3 (FGFR3), with the majority of cases resulting from a heterozygous p.Asn540Lys gain-of-function mutation. Here, we report the generation and characterization of the first mouse model (Fgfr3Asn534Lys/+) of HCH to our knowledge. Fgfr3Asn534Lys/+ mice exhibited progressive dwarfism and impairment of the synchondroses of the cranial base, resulting in defective formation of the foramen magnum. The appendicular and axial skeletons were both severely affected and we demonstrated an important role of FGFR3 in regulation of cortical and trabecular bone structure. Trabecular bone mineral density (BMD) of long bones and vertebral bodies was decreased, but cortical BMD increased with age in both tibiae and femurs. These results demonstrate that bones in Fgfr3Asn534Lys/+ mice, due to FGFR3 activation, exhibit some characteristics of osteoporosis. The present findings emphasize the detrimental effect of gain-of-function mutations in the Fgfr3 gene on long bone modeling during both developmental and aging processes, with potential implications for the management of elderly patients with hypochondroplasia and osteoporosis.
Léa Loisay, Davide Komla-Ebri, Anne Morice, Yann Heuzé, Camille Viaut, Amélie de La Seiglière, Nabil Kaci, Danny Chan, Audrey Lamouroux, Geneviève Baujat, J.H. Duncan Bassett, Graham R. Williams, Laurence Legeai-Mallet
We previously reported that measles virus nucleocapsid protein (MVNP) expression in osteoclasts (OCLs) of Paget’s disease (PD) patients or targeted to the OCL lineage in transgenic (T-MVNP) mice increases IGF1 production in osteoclasts (OCL-IGF1) and develop PD-OCLs and pagetic bone lesions (PDLs). Conditional deletion of Igf1 in OCLs of T-MVNP mice fully blocked development of pagetic bone lesions (PDLs). In this manuscript we examined if osteocytes (OCys), key regulators of normal bone remodeling, contribute to PD. OCys in PDLs of patients and of T-MVNP mice expressed less sclerostin, and had increased RANKL expression compared to OCys in bones from WT mice or normal patients. To test if increased OCL-IGF1 is sufficient to induce PDLs and PD-phenotypes, we generated TRAP-Igf1 (T-Igf1) transgenic mice to characterize if increased IGF1 expression in the absence of MVNP in OCLs is sufficient to induce pagetic lesions and pagetic OCLs. We found that T-Igf1 mice at 16 months of age developed PD-OCLs, PDLs, and OCys with decreased sclerostin and increased RANKL similar to T-MVNP mice. Thus, pagetic phenotypes could be induced by OCLs expressing increased IGF1. OCL-IGF1 in turn increased RANKL production in OCys to induce PD-OCL and PDLs.
Kazuaki Miyagawa, Hirofumi Tenshin, Patrick L. Mulcrone, Jesus Delgado-Calle, Mark A. Subler, Jolene J. Windle, John M. Chirgwin, Garson David Roodman, Noriyoshi Kurihara
Pericytes are multipotent mesenchymal precursor cells that demonstrate tissue-specific properties. In this study, by comparing human adipose and periosteal-derived pericyte microarrays, we identified TIAM1 as a key regulator of cell morphology and differentiation decisions. TIAM1 represents a tissue-specific determinant between predispositions for adipocytic versus osteoblastic differentiation in human pericytes. TIAM1 overexpression promotes an adipogenic phenotype, whereas its downregulation amplifies osteogenic differentiation. These results were replicated in vivo xenograft animal model, in which TIAM1 misexpression altered bone or adipose tissue generation in an intramuscular xenograft animal model. Changes in pericyte differentiation potential induced by TIAM1 misexpression correlated with actin organization and altered cytoskeletal morphology. Small molecule inhibitors of either Rac1 or RhoA/ROCK signaling reversed TIAM1-induced morphology and differentiation in pericytes. Finally, pericytes within calcified vessels demonstrated decreased TIAM1 expression in the diseased area compared to the healthy tissue. In summary, our results demonstrate that TIAM1 regulates the cellular morphology and differentiation potential of human pericytes, representing a molecular switch between osteogenic and adipogenic cell fates.
Ginny Ching-Yun Hsu, Yiyun Wang, Amy Z. Lu, Mario A. Gomez-Salazar, Jiajia Xu, Dongqing Li, Carolyn Meyers, Stefano Negri, Sintawat Wangsiricharoen, Kristen P. Broderick, Bruno Peault, Carol D. Morris, Aaron W. James
The intake of dietary phosphate far exceeds recommended levels however the long-term health consequences remain relatively unknown. Here, the chronic physiological response to sustained elevated and reduced dietary phosphate consumption was investigated in mice. Although serum phosphate levels were brought into homeostatic balance, the prolonged intake of a high-phosphate diet dramatically and negatively impacted bone volume, generated a sustained increase in the phosphate responsive circulating factors, FGF23, PTH, osteopontin and osteocalcin, and produced a chronic low grade inflammatory state in the bone marrow, marked by increased numbers of T cells expressing IL-17a, RANKL, and TNFα. In contrast, a low-phosphate diet preserved trabecular bone while increasing cortical bone volume over time and reduced inflammatory T cell populations. Cell-based studies identified a direct response of T cells to elevated extracellular phosphate. Neutralizing antibodies to pro-osteoclastic cytokines RANKL, TNFα, and IL-17a blunted the high-phosphate diet induced bone loss identifying bone resorption as a regulatory mechanism. Collectively, this study illuminates that habitual consumption of a high-phosphate diet in mice induces chronic inflammation in bone even in the absence of elevated serum phosphate. Further, the study supports the concept that a reduced phosphate diet may be a simple, yet effective strategy to reduce inflammation and improve bone health during aging
Joseph L. Roberts, Mingcan Yu, Manjula Viggeswarapu, Jamie L. Arnst, Roberto Pacifici, George R. Beck
Glycolysis is central to homeostasis of nucleus pulposus (NP) cells in the avascular intervertebral disc. Since the glucose importer, GLUT1, is a highly enriched phenotypic marker of NP cells, we hypothesized that it is vital for the development and post-natal maintenance of the disc. Surprisingly, primary NP cells treated with two well-characterized GLUT1 inhibitors maintained normal rates of glycolysis and ATP production, indicating intrinsic compensatory mechanisms. We show in vitro that NP cells mitigate GLUT1 loss by rewiring glucose import through GLUT3. Noteworthy, we demonstrate that substrates, such as glutamine and palmitate, do not compensate for glucose restriction resulting from dual inhibition of GLUT1/3 and inhibition compromises long-term cell viability. To investigate the redundancy of GLUT1 function in NP, we generated two NP-specific knockout mice: Krt19CreERT; Glut1f/f and Foxa2Cre; Glut1f/f. Noteworthy, there were no apparent defects in post-natal disc health or development and maturation in mutant mice. Microarray analysis confirmed that GLUT1 loss did not cause transcriptomic alterations in the NP, supporting that cells are refractory to GLUT1 loss. These observations provide the first evidence of functional redundancy in GLUT transporters in the physiologically hypoxic intervertebral disc and underscore the importance of glucose as the indispensable substrate for NP cells.
Shira N. Johnston, Elizabeth S. Silagi, Vedavathi Madhu, Duc H. Nguyen, Irving M. Shapiro, Makarand V. Risbud
Hypothalamic neurons regulate body homeostasis by sensing and integrating changes in the levels of key hormones and primary nutrients (amino acids, glucose, and lipids). However, the molecular mechanisms that enable hypothalamic neurons to detect primary nutrients remain elusive. Here, we identified L-type amino acid transporter 1 (LAT1) in hypothalamic leptin receptor (LepR)-expressing neurons as being important for systemic energy and bone homeostasis. We observed LAT1-dependent amino acid uptake in the hypothalamus, which was compromised in a mouse model of obesity and diabetes. Mice lacking LAT1 (encoded by Slc7a5) in LepR-expressing neurons exhibited obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR-expressing neurons before obesity onset. Importantly, restoring Slc7a5 expression selectively in LepR-expressing ventromedial hypothalamus neurons rescued energy and bone homeostasis in mice deficient for Slc7a5 in LepR-expressing cells. Mechanistic target of rapamycin complex-1 (mTORC1) was found to be a crucial mediator of LAT1-dependent regulation of energy and bone homeostasis. These results suggest that the LAT1–mTORC1 axis in LepR-expressing neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow, thus providing in vivo evidence of the implications of amino acid sensing by hypothalamic neurons in body homeostasis.
Gyujin Park, Kazuya Fukasawa, Tetsuhiro Horie, Yusuke Masuo, Yuka Inaba, Takanori Tatsuno, Takanori Yamada, Kazuya Tokumura, Sayuki Iwahashi, Takashi Iezaki, Katsuyuki Kaneda, Yukio Kato, Yasuhito Ishigaki, Michihiro Mieda, Tomohiro Tanaka, Kazuma Ogawa, Hiroki Ochi, Shingo Sato, Yun-Bo Shi, Hiroshi Inoue, Hojoon Lee, Eiichi Hinoi
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