RESUMEN
Heterotopic ossification is the most disabling feature of fibrodysplasia ossificans progressiva, an ultra-rare genetic disorder for which there is currently no prevention or treatment. Most patients with this disease harbor a heterozygous activating mutation (c.617 G > A;p.R206H) in ACVR1. Here, we identify recombinant AAV9 as the most effective serotype for transduction of the major cells-of-origin of heterotopic ossification. We use AAV9 delivery for gene replacement by expression of codon-optimized human ACVR1, ACVR1R206H allele-specific silencing by AAV-compatible artificial miRNA and a combination of gene replacement and silencing. In mouse skeletal cells harboring a conditional knock-in allele of human mutant ACVR1 and in patient-derived induced pluripotent stem cells, AAV gene therapy ablated aberrant Activin A signaling and chondrogenic and osteogenic differentiation. In Acvr1(R206H) knock-in mice treated locally in early adulthood or systemically at birth, trauma-induced endochondral bone formation was markedly reduced, while inflammation and fibroproliferative responses remained largely intact in the injured muscle. Remarkably, spontaneous heterotopic ossification also substantially decreased in in Acvr1(R206H) knock-in mice treated systemically at birth or in early adulthood. Collectively, we develop promising gene therapeutics that can prevent disabling heterotopic ossification in mice, supporting clinical translation to patients with fibrodysplasia ossificans progressiva.
Asunto(s)
MicroARNs , Miositis Osificante , Osificación Heterotópica , Adulto , Animales , Humanos , Ratones , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Terapia Genética , Ratones Transgénicos , Mutación , Miositis Osificante/genética , Miositis Osificante/terapia , Osificación Heterotópica/genética , Osificación Heterotópica/terapia , Osificación Heterotópica/metabolismo , Osteogénesis/genética , Adenoviridae/genéticaRESUMEN
Osteocytes play a critical role in bone remodeling through the secretion of paracrine factors regulating the differentiation and activity of osteoblasts and osteoclasts. Sclerostin is a key osteocyte-derived factor that suppresses bone formation and promotes bone resorption, therefore regulators of sclerostin secretion are a likely source of new therapeutic strategies for treatment of skeletal disorders. Here, we demonstrate that protein kinase CK2 (casein kinase 2) controls sclerostin expression in osteocytes via the deubiquitinase ubiquitin-specific peptidase 4 (USP4)-mediated stabilization of Sirtuin1 (SIRT1). Deletion of CK2 regulatory subunit, Csnk2b, in osteocytes (Csnk2bDmp1) results in low bone mass due to elevated levels of sclerostin. This phenotype in Csnk2bDmp1 mice was partly reversed when sclerostin expression was downregulated by a single intravenous injection with bone-targeting adeno-associated virus 9 (AAV9) carrying an artificial-microRNA that targets Sost. Mechanistically, CK2-induced phosphorylation of USP4 is important for stabilization of SIRT1 by suppressing ubiquitin-dependent proteasomal degradation. Upregulated expression of SIRT1 inhibits sclerostin transcription in osteocytes. Collectively, the CK2-USP4-SIRT1 pathway is crucial for the regulation of sclerostin expression in osteocytes to maintain bone homeostasis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Osteocitos , Sirtuina 1 , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Ratones , Osteoblastos/metabolismo , Osteocitos/metabolismo , Osteogénesis , Sirtuina 1/metabolismoRESUMEN
Emerging evidence supports that osteogenic differentiation of skeletal progenitors is a key determinant of overall bone formation and bone mass. Despite extensive studies showing the function of mitogen-activated protein kinases (MAPKs) in osteoblast differentiation, none of these studies show in vivo evidence of a role for MAPKs in osteoblast maturation subsequent to lineage commitment. Here, we describe how the extracellular signal-regulated kinase (ERK) pathway in osteoblasts controls bone formation by suppressing the mechanistic target of rapamycin (mTOR) pathway. We also show that, while ERK inhibition blocks the differentiation of osteogenic precursors when initiated at an early stage, ERK inhibition surprisingly promotes the later stages of osteoblast differentiation. Accordingly, inhibition of the ERK pathway using a small compound inhibitor or conditional deletion of the MAP2Ks Map2k1 (MEK1) and Map2k2 (MEK2), in mature osteoblasts and osteocytes, markedly increased bone formation due to augmented osteoblast differentiation. Mice with inducible deletion of the ERK pathway in mature osteoblasts also displayed similar phenotypes, demonstrating that this phenotype reflects continuous postnatal inhibition of late-stage osteoblast maturation. Mechanistically, ERK inhibition increases mitochondrial function and SGK1 phosphorylation via mTOR2 activation, which leads to osteoblast differentiation and production of angiogenic and osteogenic factors to promote bone formation. This phenotype was partially reversed by inhibiting mTOR. Our study uncovers a surprising dichotomy of ERK pathway functions in osteoblasts, whereby ERK activation promotes the early differentiation of osteoblast precursors, but inhibits the subsequent differentiation of committed osteoblasts via mTOR-mediated regulation of mitochondrial function and SGK1.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular , Osteogénesis , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Osteoblastos/metabolismo , Fosforilación , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Shift workers and many other groups experience irregular sleep-wake patterns. This can induce excessive daytime sleepiness that decreases productivity and elevates the risk of accidents. However, the degree of daytime sleepiness is not correlated with standard sleep parameters like total sleep time, suggesting other factors are involved. Here, we analyze real-world sleep-wake patterns of shift workers measured with wearables by developing a computational package that simulates homeostatic sleep pressure - physiological need for sleep - and the circadian rhythm. This reveals that shift workers who align sleep-wake patterns with their circadian rhythm have lower daytime sleepiness, even if they sleep less. The alignment, quantified by the sleep parameter, circadian sleep sufficiency, can be increased by dynamically adjusting daily sleep durations according to varying bedtimes. Our computational package provides flexible and personalized real-time sleep-wake patterns for individuals to reduce their daytime sleepiness and could be used with wearables to develop smart alarms.