Comprehensive analysis of the proximity-dependent nuclear interactome for the oncoprotein NOTCH1 in live cells.

Notch signaling plays a critical role in cell fate decisions in all cell types. Furthermore, gain-of-function mutations in NOTCH1 have been uncovered in many human cancers. Disruption of Notch signaling has recently emerged as an attractive disease treatment strategy. However, the nuclear interaction landscape of the oncoprotein NOTCH1 remains largely unexplored. We therefore employed here a proximity-dependent biotin identification approach to identify in vivo protein associations with the nuclear Notch1 intracellular domain in live cells. We identified a large set of previously reported and unreported proteins that associate with NOTCH1, including general transcription and elongation factors, DNA repair and replication factors, coactivators, corepressors, and components of the NuRD and SWI/SNF chromatin remodeling complexes. We also found that Notch1 intracellular domain associates with protein modifiers and components of other signaling pathways that may influence Notch signal transduction and protein stability such as USP7. We further validated the interaction of NOTCH1 with histone deacetylase 1 or GATAD2B using protein network analysis, proximity-based ligation, in vivo cross-linking and coimmunoprecipitation assays in several Notch-addicted cancer cell lines. Through data mining, we also revealed potential drug targets for the inhibition of Notch signaling. Collectively, these results provide a valuable resource to uncover the mechanisms that fine-tune Notch signaling in tumorigenesis and inform therapeutic targets for Notch-addicted tumors.

Hdac1 and Hdac2 positively regulate Notch1 gain-of-function pathogenic signaling in committed osteoblasts of male mice.

BACKGROUND: Skeletal development requires precise extrinsic and intrinsic signals to regulate processes that form and maintain bone and cartilage. Notch1 is a highly conserved signaling receptor that regulates cell fate decisions by controlling the duration of transcriptional bursts. Epigenetic molecular events reversibly modify DNA and histone tails by influencing the spatial organization of chromatin and can fine-tune the outcome of a Notch1 transcriptional response. Histone deacetylase 1 and 2 (HDAC1 and HDAC2) are chromatin modifying enzymes that mediate osteoblast differentiation. While an HDAC1-Notch interaction has been studied in vitro and in Drosophila, its role in mammalian skeletal development and disorders is unclear. Osteosclerosis is a bone disorder with an abnormal increase in the number of osteoblasts and excessive bone formation. METHODS: Here, we tested whether Hdac1/2 contribute to the pathogenesis of osteosclerosis in a murine model of the disease owing to conditionally cre-activated expression of the Notch1 intracellular domain in immature osteoblasts. RESULTS: Importantly, selective homozygous deletions of Hdac1/2 in osteoblasts partially alleviate osteosclerotic phenotypes (Col2.3kb-Cre; TGRosaN1ICD/+ ; Hdac1flox/flox ; Hdac2flox/flox ) with a 40% decrease in bone volume and a 22% decrease in trabecular thickness in 4 weeks old when compared to male mice with heterozygous deletions of Hdac1/2 (Col2.3 kb-Cre; TGRosaN1ICD/+ ; Hdac1flox/+ ; Hdac2flox/+ ). Osteoblast-specific deletion of Hdac1/2 in male and female mice results in no overt bone phenotype in the absence of the Notch1 gain-of-function (GOF) allele. CONCLUSIONS: These results provide evidence that Hdac1/2 contribute to Notch1 pathogenic signaling in the mammalian skeleton. Our study on epigenetic regulation of Notch1 GOF-induced osteosclerosis may facilitate further mechanistic studies of skeletal birth defects caused by Notch-related GOF mutations in human patients, such as Adams-Oliver disease, congenital heart disease, and lateral meningocele syndrome.

Pulling rank with RNA: RANKL promotes the association of PGC-1ß/RNA complexes with NCoR/HDAC3 to activate gene expression in osteoclasts.

In this issue, Abe et al1 report a novel mechanism by which RANKL stimulates osteoclast differentiation and bone resorption through non-coding RNAs that bind PGC-1ß and convert the NCoR/HDAC3 co-repressor complex into a co-activator of AP-1- and NFκB-regulated genes.

Inflammatory Processes Affecting Bone Health and Repair.

PURPOSE OF REVIEW: The purpose of this article is to review the current understanding of inflammatory processes on bone, including direct impacts of inflammatory factors on bone cells, the effect of senescence on inflamed bone, and the critical role of inflammation in bone pain and healing. RECENT FINDINGS: Advances in osteoimmunology have provided new perspectives on inflammatory bone loss in recent years. Characterization of so-called inflammatory osteoclasts has revealed insights into physiological and pathological bone loss. The identification of inflammation-associated senescent markers in bone cells indicates that therapies that reduce senescent cell burden may reverse bone loss caused by inflammatory processes. Finally, novel studies have refined the role of inflammation in bone healing, including cross talk between nerves and bone cells. Except for the initial stages of fracture healing, inflammation has predominately negative effects on bone and increases fracture risk. Eliminating senescent cells, priming the osteo-immune axis in bone cells, and alleviating pro-inflammatory cytokine burden may ameliorate the negative effects of inflammation on bone.

Selective Targeting of Class I Histone Deacetylases in a Model of Human Osteosarcoma.

Dysregulation of histone deacetylases (HDACs) is associated with the pathogenesis of human osteosarcoma, which may present an epigenetic vulnerability as well as a therapeutic target. Domatinostat (4SC-202) is a next-generation class I HDAC inhibitor that is currently being used in clinical research for certain cancers, but its impact on human osteosarcoma has yet to be explored. In this study, we report that 4SC-202 inhibits osteosarcoma cell growth in vitro and in vivo. By analyzing cell function in vitro, we show that the anti-tumor effect of 4SC-202 involves the combined induction of cell-cycle arrest at the G2/M phase and apoptotic program, as well as a reduction in cell invasion and migration capabilities. We also found that 4SC-202 has little capacity to promote osteogenic differentiation. Remarkably, 4SC-202 revised the global transcriptome and induced distinct signatures of gene expression in vitro. Moreover, 4SC-202 decreased tumor growth of established human tumor xenografts in immunodeficient mice in vivo. We further reveal key targets regulated by 4SC-202 that contribute to tumor cell growth and survival, and canonical signaling pathways associated with progression and metastasis of osteosarcoma. Our study suggests that 4SC-202 may be exploited as a valuable drug to promote more effective treatment of patients with osteosarcoma and provide molecular insights into the mechanism of action of class I HDAC inhibitors.

Precise detection of a murine germline mutation of the Notch3 gene associated with kyphosis and developmental disorders.

OBJECTIVE: Humpback (hpbk) mice harbor a pathogenic mutation in the Notch3 gene and can serve as a beneficial animal model for investigating human myopathy, kyphosis, and developmental disorders, including lateral meningocele syndrome. Detection of the point mutation in hpbk mice is important for maintaining strains and scrutinizing genetic rescues, especially considering that homozygous mice are infertile and indistinguishable from their littermates at a young age. This study aimed for the development of a novel, precise, and time-saving genotyping method to identify the mutation in hpbk mice. MATERIALS AND METHODS: In order to study the hpbk mouse line, we describe how we applied several tools, including quantitative polymerase chain reaction (qPCR), multiplex tetra-primer amplification-refractory mutation system (ARMS-PCR) and Sanger sequencing, toward the recognition of heterozygous and homozygous mice. RESULTS: The Notch3 mutation was clearly identified using qPCR and ARMS assays, but the latter was a more precise and cost-effective approach. The lengths of the ARMS-PCR amplicons are 210 bp and 164 bp for the wild-type and hpbk alleles, respectively. Moreover, the genotyping results for each mouse were corroborated by Sanger DNA sequencing. CONCLUSION: Our newly developed PCR-based ARMS system affords a swift and precise way to genotype the hpbk mice. ARMS-PCR does not rely on any advanced equipment and is useful as a genotyping method for other model organisms that harbor a pathogenic variant.

Statin-mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration.

KEY POINTS: This study demonstrates and evaluates the changes in rat vascular smooth muscle cell biomechanics following statin-mediated cholesterol depletion. Evidence is presented to show correlated changes in migration and adhesion of vascular smooth muscle cells to extracellular matrix proteins fibronectin and collagen. Concurrently, integrin α5 expression was enhanced but not integrin α2. Atomic force microscopy analysis provides compelling evidence of coordinated reduction in vascular smooth muscle cell stiffness and actin cytoskeletal orientation in response to statin-mediated cholesterol depletion. Proof is provided that statin-mediated cholesterol depletion remodels total vascular smooth muscle cell cytoskeletal orientation that may additionally participate in altering ex vivo aortic vessel function. It is concluded that statin-mediated cholesterol depletion may coordinate vascular smooth muscle cell migration and adhesion to different extracellular matrix proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell. ABSTRACT: Not only does cholesterol induce an inflammatory response and deposits in foam cells at the atherosclerotic plaque, it also regulates cellular mechanics, proliferation and migration in atherosclerosis progression. Statins are HMG-CoA reductase inhibitors that are known to inhibit cellular cholesterol biosynthesis and are clinically prescribed to patients with hypercholesterolemia or related cardiovascular conditions. Nonetheless, the effect of statin-mediated cholesterol management on cellular biomechanics is not fully understood. In this study, we aimed to assess the effect of fluvastatin-mediated cholesterol management on primary rat vascular smooth muscle cell (VSMC) biomechanics. Real-time measurement of cell adhesion, stiffness, and imaging were performed using atomic force microscopy (AFM). Cellular migration on extra cellular matrix (ECM) protein surfaces was studied by time-lapse imaging. The effect of changes in VSMC biomechanics on aortic function was assessed using an ex vivo myograph system. Fluvastatin-mediated cholesterol depletion (-27.8%) lowered VSMC migration distance on a fibronectin (FN)-coated surface (-14.8%) but not on a type 1 collagen (COL1)-coated surface. VSMC adhesion force to FN (+33%) and integrin α5 expression were enhanced but COL1 adhesion and integrin α2 expression were unchanged upon cholesterol depletion. In addition, VSMC stiffness (-46.6%) and ex vivo aortic ring contraction force (-40.1%) were lowered and VSMC actin cytoskeletal orientation was reduced (-24.5%) following statin-mediated cholesterol depletion. Altogether, it is concluded that statin-mediated cholesterol depletion may coordinate VSMC migration and adhesion to different ECM proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell and aortic function.

FAK tyrosine 407 organized with integrin αVß5 in Hs578Ts(i)8 advanced triple-negative breast cancer cells.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase known to promote cell migration and invasiveness. Overexpression and increased activity of FAK are closely associated with metastatic breast tumors and are linked to poor prognosis. This study discovered an inverse correlation between FAK activity and migratory and invasive behavior. We show decreased phosphorylation levels of FAK at tyrosine residues 397 and 861, and most prominently at Y407, in the more invasive Hs578Ts(i)8 subclone of the Hs578T breast cancer progression model. There is limited information available on FAK Y407, and here we demonstrate its presence in triple-negative breast cancer (TNBC) cell lines. Furthermore, our studies propose that localization of FAK Y407, rather than FAK expression and overall FAK Y407 phosphorylation levels, is crucial for the control of cell motility. FAK Y407 is found extensively at the cell periphery in focal adhesion-like structures at each end of actin stress fibers and organized with integrin αVß5 receptors, linking the αVß5 integrin-mediated migratory behavior of Hs578Ts(i)8 cells to FAK Y407. These data suggest that subcellular localization, next to expression and activity levels, are important for understanding TNBC progression. Such an approach opens new avenues for further studies and may provide novel insight for the classification of TNBC and facilitate the discovery of effective biomarkers for diagnosis and therapy of TNBC.