Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, ...organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development.
Osteoarthritis (OA) is a common degenerative condition that afflicts more than 70% of the population between 55 and 77 years of age. Although its prevalence is rising globally with aging of the ...population, current therapy is limited to symptomatic relief and, in severe cases, joint replacement surgery. We report that intra-articular expression of proteoglycan 4 (Prg4) in mice protects against development of OA. Long-term Prg4 expression under the type II collagen promoter (Col2a1) does not adversely affect skeletal development but protects from developing signs of age-related OA. The protective effect is also shown in a model of posttraumatic OA created by cruciate ligament transection. Moreover, intra-articular injection of helper-dependent adenoviral vector expressing Prg4 protected against the development of posttraumatic OA when administered either before or after injury. Gene expression profiling of mouse articular cartilage and in vitro cell studies show that Prg4 expression inhibits the transcriptional programs that promote cartilage catabolism and hypertrophy through the up-regulation of hypoxia-inducible factor 3α. Analyses of available human OA data sets are consistent with the predictions of this model. Hence, our data provide insight into the mechanisms for OA development and offer a potential chondroprotective approach to its treatment.
Objective
The mouse is an optimal model organism in which gene–environment interactions can be used to study the pathogenesis of osteoarthritis (OA). The gold standard for arthritis research in mice ...is based on histopathology and immunohistochemistry, which are labor‐intensive, prone to sampling bias and technical variability, and limited in throughput. The aim of this study was to develop a new technique that assesses mouse cartilage by integrating quantitative volumetric imaging techniques.
Methods
A novel mouse model of OA was generated by cruciate ligament transection (CLT) and evaluated by histopathology and immunohistochemistry. Knee joint specimens were then imaged using a new technique that combines high‐resolution micro–computed tomography (micro‐CT) and phase‐contrast optics followed by quantitative analyses. A comparative analysis was also performed in a previously established mouse model of OA generated by destabilization of the medial meniscus (DMM).
Results
Phase‐contrast micro‐CT achieved cellular resolution of chondrocytes and quantitative assessment of parameters such as articular cartilage volume and surface area. In mouse models of OA generated by either CLT or DMM, we showed that phase‐contrast micro‐CT distinguished control and OA cartilage by providing quantitative measures with high reproducibility and minimal variability. Features of OA at the cellular or tissue level could also be observed in images generated by phase‐contrast micro‐CT.
Conclusion
We established an imaging technology that comprehensively assessed and quantified the 2‐dimensional and 3‐dimensional changes of articular cartilage. Application of this technology will facilitate the rapid and high‐throughput assessment of genetic and therapeutic models of OA in mice.
Mutations in WNT1 cause osteogenesis imperfecta (OI) and early-onset osteoporosis, identifying it as a key Wnt ligand in human bone homeostasis. However, how and where WNT1 acts in bone are unclear. ...To address this mechanism, we generated late-osteoblast-specific and osteocyte-specific WNT1 loss- and gain-of-function mouse models. Deletion of Wnt1 in osteocytes resulted in low bone mass with spontaneous fractures similar to that observed in OI patients. Conversely, Wnt1 overexpression from osteocytes stimulated bone formation by increasing osteoblast number and activity, which was due in part to activation of mTORC1 signaling. While antiresorptive therapy is the mainstay of OI treatment, it has limited efficacy in WNT1-related OI. In this study, anti-sclerostin antibody (Scl-Ab) treatment effectively improved bone mass and dramatically decreased fracture rate in swaying mice, a model of global Wnt1 loss. Collectively, our data suggest that WNT1-related OI and osteoporosis are caused in part by decreased mTORC1-dependent osteoblast function resulting from loss of WNT1 signaling in osteocytes. As such, this work identifies an anabolic function of osteocytes as a source of Wnt in bone development and homoeostasis, complementing their known function as targets of Wnt signaling in regulating osteoclastogenesis. Finally, this study suggests that Scl-Ab is an effective genotype-specific treatment option for WNT1-related OI and osteoporosis.
•Gene therapy uses vectors to deliver therapeutic nucleic acids to diseased tissues.•Classic approaches target inflammation and tissue remodeling to promote repair.•Monotherapies targeting IL-1Ra, ...TGF-β and others are being evaluated for OA and RA.•Combinatorial and inducible strategies are emerging to address diseases complexity.•miRNAs may offer a means to modulate multiple targets with a single therapeutic.
Gene therapy refers to the use of viral and non-viral vectors to deliver nucleic acids to tissues of interest using direct (in vivo) or transduced cell-mediated (ex vivo) approaches. Over the past few decades, strategies have been adopted to express therapeutic transgenes at sites of injury to promote or facilitate repair of bone and cartilage. Targets of interest have typically included secreted proteins such as growth factors and anti-inflammatory mediators; however, work has also begun to focus intracellularly on signaling components, transcription factors and small, regulatory nucleic acids such as microRNAs (miRNAs). In recent years, a number of single therapeutic gene approaches (termed ‘monotherapies’) have proven effective in preclinical models of disease, and several are being evaluated in clinical trials. In particular, an ex vivo TGF-β1 gene therapy was approved in Korea in 2017 for treatment of moderate-to-severe osteoarthritis (OA). The ability to utilize viral vectors for context-specific and combinatorial gene therapy is also being investigated, and these strategies are likely to be important in more robustly addressing the complexities of tissue repair and regeneration in skeletal disease. In this review, we provide an overview of viral gene therapies being developed for treatment of bone and cartilage pathologies, with an emphasis on emerging combinatorial strategies as well as those targeting intracellular mediators such as miRNAs.
Osteogenic sarcoma (OS) is a deadly skeletal malignancy whose cause is unknown. We report here a mouse model of OS based on conditional expression of the intracellular domain of Notch1 (NICD). ...Expression of the NICD in immature osteoblasts was sufficient to drive the formation of bone tumors, including OS, with complete penetrance. These tumors display features of human OS; namely, histopathology, cytogenetic complexity, and metastatic potential. We show that Notch activation combined with loss of p53 synergistically accelerates OS development in mice, although p53-driven OS is not Rbpj dependent, which demonstrates a dual dominance of the Notch oncogene and p53 mutation in the development of OS. Using this model, we also reveal the osteoblasts as the potential sources of OS.
•Notch 1 intracellular domain induces bone tumors in mice with 100% penetrance•Mouse osteosarcoma initiated by Notch activation mimics the human disease•Notch activation and p53 loss independently and synergistically induce osteosarcoma•Committed osteoblasts are a potential source of tumor cells in osteosarcomagenesis
Tao et al. show that Notch activation in osteoblasts is sufficient to initiate osteogenic sarcoma (OS)-like tumors that mimic human OS and demonstrate a dual dominance for Notch activation and p53 mutation in the development of OS.
Osteocytes are the terminally differentiated cell type of the osteoblastic lineage and have important functions in skeletal homeostasis. Although the transcriptional regulation of osteoblast ...differentiation has been well characterized, the factors that regulate differentiation of osteocytes from mature osteoblasts are poorly understood. Here we show that miR-23a∼27a∼24-2 (miR-23a cluster) promotes osteocyte differentiation. Osteoblast-specific miR-23a cluster gain-of-function mice have low bone mass associated with decreased osteoblast but increased osteocyte numbers. By contrast, loss-of-function transgenic mice overexpressing microRNA decoys for either miR-23a or miR-27a, but not miR24-2, show decreased osteocyte numbers. Moreover, RNA-sequencing analysis shows altered transforming growth factor-β (TGF-β) signalling. Prdm16, a negative regulator of the TGF-β pathway, is directly repressed by miR-27a with concomitant alteration of sclerostin expression, and pharmacological inhibition of TGF-β rescues the phenotypes observed in the gain-of-function transgenic mice. Taken together, the miR-23a cluster regulates osteocyte differentiation by modulating the TGF-β signalling pathway through targeting of Prdm16.
Objective
Gene therapy holds great promise for the treatment of osteoarthritis (OA) because a single intraarticular injection can lead to long‐term expression of therapeutic proteins within the ...joint. This study was undertaken to investigate the use of a helper‐dependent adenovirus (HDAd)–mediated intraarticular gene therapy approach for long‐term expression of interleukin‐1 receptor antagonist (IL‐1Ra) as sustained symptomatic and disease‐modifying therapy for OA.
Methods
In mouse models of OA, efficacy of HDAd–IL‐1Ra was evaluated by histologic analysis, micro–computed tomography (micro‐CT), and hot plate analysis. In a horse OA model, safety and efficacy of HDAd–IL‐1Ra were evaluated by blood chemistry, analyses of synovial fluid, synovial membrane, and cartilage, and gross pathology and lameness assessments.
Results
In skeletally immature mice, HDAd–IL‐1Ra prevented development of cartilage damage, osteophytes, and synovitis. In skeletally immature and mature mice, treatment with HDAd–interleukin‐1 receptor antagonist post–OA induction resulted in improved—albeit not significantly—cartilage status assessed histologically and significantly increased cartilage volume, cartilage surface, and bone surface covered by cartilage as assessed by micro‐CT. Fewer osteophytes were observed in HDAd–IL‐1Ra–treated skeletally immature mice. Synovitis was not affected in skeletally immature or mature mice. HDAd–IL‐1Ra protected against disease‐induced thermal hyperalgesia in skeletally mature mice. In the horse OA model, HDAd–IL‐1Ra therapy significantly improved lameness parameters, indicating efficient symptomatic treatment. Moreover, macroscopically and histologically assessed cartilage and synovial membrane parameters were significantly improved, suggesting disease‐modifying efficacy.
Conclusion
These data from OA models in small and large animals demonstrated safe symptomatic and disease‐modifying treatment with an HDAd‐expressing IL‐1Ra. Furthermore, this study establishes HDAd as a vector for joint gene therapy.
A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD precursors reverse ...aspects of aging, in part, by activating sirtuin deacylases (SIRT1–SIRT7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ booster nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly.
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•Reduced blood flow with age is due to loss of endothelial NAD+-SIRT1 activity•NAD+ and H2S control muscle angiogenesis and increase endurance in old mice•The NAD precursor NMN mimics and augments exercise by inhibiting NICD-Notch•Neovascularization is as important as mitochondria for rejuvenating muscle
Endothelial SIRT1 regulates pro-angiogenic signals secreted from myocytes and improves muscle health. Treatment of mice with NAD precursor nicotinamide mononucleotide improves vascular and increases endurance in aging mice.
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