Project description:Paget’s disease of bone (PDB) is a chronic focal skeletal disorder that affects 2-3% of the population over the age of 60. PDB is inherited as an autosomal dominant trait with genetic heterogeneity. SQSTM1/p62 UBA domain mutation (p62P392L) is widely identified in PDB and has been shown to increase osteoclastogenesis. Further, environmental factors such as paramyxovirus are implicated in PDB and MVNP has been shown to induce Pagetic phenotype in osteoclasts. However, the molecular mechanisms underlying p62P392L and MVNP stimulation of osteoclast differentiation in PDB are unclear. We therefore determined MVNP regulated gene expression profiling during osteoclast differentiation. We identified 8.4% of genes were upregulated (> 4-fold) in MVNP transduced cells. MVNP increased integrin β3 (63 fold), NFAT activating protein (6.5 fold), OSCAR (5.5 fold), TRAF5 (8.5 fold) mRNA expression compared to empty vector (EV) transduced cells. MVNP also elevated gene expression of cytokines/growth factors such as IL-17 (18 fold), IL-1F7 (10 fold), IL-17R (4.5 fold) and IL-11 (7 fold). Interestingly, MVNP transduced cells demonstrated high level expression of signal regulatory protein beta 1 (SIRPβ1) (353 fold). SIRPβ1 has been shown to interact with DAP 12, an ITAM containing adaptor protein which plays an important role in osteoclast differentiation. Real-time PCR analysis of total RNA isolated from normal human peripheral blood monocytes transduced with MVNP confirmed upregulation of SIRPβ1 mRNA expression in the absence of RANKL stimulation. In contrast, RANKL stimulation did not alter SIRPβ1 expression in these cells. Furthermore, bone marrow mononuclear cells derived from patients with PDB showed high levels of SIRPβ1 mRNA expression compared to normal subjects. Thus, MVNP regulated gene expression profiling during osteoclast differentiation provides new insights into molecular mechanisms and therapeutic targets to control elevated osteoclast activity in PDB. Total RNA isolated from normal human bone marrow mononuclear cells transduced with EV, MVNP retroviral expression vectors and stimulated with M-CSF and RANKL for 48 h were subjected to Agilent microarray (~26,000 genes) analysis. One replicate per treatment was hybridized.

Project description:Lifestyle disorders like obesity, type 2 diabetes (T2D), and cardiovascular diseases can be prevented and treated by regular physical activity. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24 h to human myotubes isolated and differentiated from biopsy samples from 6 morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24 h thereafter. Size and concentration of EV subtypes were characterized by nanoparticle tracking analysis, surface markers were examined by flow cytometry and Western blotting, and morphology was confirmed by transmission electron microscopy. Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix microarray, and selected microRNAs (miRs) validated by real time RT-qPCR. The size and concentration of exosomes and MV were unaffected by EPS. Of the 400 miRs identified in the EVs, EPS significantly changed the level of 15 exosome miRs, of which miR-1233-5p showed the highest fold change. The miR pattern of MV was unaffected by EPS. Totally, about 1000 proteins were identified in exosomes and 2000 in MV. EPS changed the content of 73 proteins in exosomes, 97 in MVs, and of these 4 were changed in both exosomes and MV (GANAB, HSPA9, CNDP2, and ATP5B). By matching the EPS-changed miRs and proteins in exosomes, 31 targets were identified, and among these several promising signaling factors. Of particular interest were CNDP2, an enzyme that generates the appetite regulatory metabolite Lac-Phe, and miR-4433b-3p, which targets CNDP2. Several of the regulated miRs, such as miR-92b-5p, miR-320b, and miR-1233-5p might also mediate interesting signaling functions.

Project description:Paget’s disease of bone (PDB) is a chronic focal skeletal disorder that affects 2-3% of the population over the age of 60. PDB is inherited as an autosomal dominant trait with genetic heterogeneity. SQSTM1/p62 UBA domain mutation (p62P392L) is widely identified in PDB and has been shown to increase osteoclastogenesis. Further, environmental factors such as paramyxovirus are implicated in PDB and MVNP has been shown to induce Pagetic phenotype in osteoclasts. However, the molecular mechanisms underlying p62P392L and MVNP stimulation of osteoclast differentiation in PDB are unclear. We therefore determined MVNP regulated gene expression profiling during osteoclast differentiation. We identified 8.4% of genes were upregulated (> 4-fold) in MVNP transduced cells. MVNP increased integrin β3 (63 fold), NFAT activating protein (6.5 fold), OSCAR (5.5 fold), TRAF5 (8.5 fold) mRNA expression compared to empty vector (EV) transduced cells. MVNP also elevated gene expression of cytokines/growth factors such as IL-17 (18 fold), IL-1F7 (10 fold), IL-17R (4.5 fold) and IL-11 (7 fold). Interestingly, MVNP transduced cells demonstrated high level expression of signal regulatory protein beta 1 (SIRPβ1) (353 fold). SIRPβ1 has been shown to interact with DAP 12, an ITAM containing adaptor protein which plays an important role in osteoclast differentiation. Real-time PCR analysis of total RNA isolated from normal human peripheral blood monocytes transduced with MVNP confirmed upregulation of SIRPβ1 mRNA expression in the absence of RANKL stimulation. In contrast, RANKL stimulation did not alter SIRPβ1 expression in these cells. Furthermore, bone marrow mononuclear cells derived from patients with PDB showed high levels of SIRPβ1 mRNA expression compared to normal subjects. Thus, MVNP regulated gene expression profiling during osteoclast differentiation provides new insights into molecular mechanisms and therapeutic targets to control elevated osteoclast activity in PDB.

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MiR profile in pagetic osteoclasts: from large-scale sequencing to gene expression study

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