Notch signaling pathways in human thoracic ossification of the ligamentum flavum

Abstract

This study investigated the pathological process of Notch signaling in the osteogenesis of ligamentum flavum tissues and cells, and the associated regulatory mechanisms. Notch receptors, ligands, and target genes were identified by quantitative polymerase chain reaction (qPCR) in ligamentum flavum cells and immunohistochemistry in ligamentum flavum sections from ossification of the ligamentum flavum (OLF) patients and controls. The temporospatial expression patterns of JAG1/Notch2/HES1 in human ligamentum flavum cells during osteogenic differentiation were determined by qPCR. Lentiviral vectors for Notch2 overexpression and knockdown were constructed and transfected into ligamentum flavum cells before osteogenic differentiation to examine the function of Notch signaling pathways in the osteogenic differentiation of ligamentum flavum cells. Alkaline phosphatase, Runx2, Osterix, osteocalcin, and osteopontin mRNA levels, alkaline phosphatase activity, and Alizarin Red staining were used as indicators of osteogenic differentiation. JAG1/Notch2/ HES1 mRNA levels were up-regulated in ligamentum flavum cells from OLF patients, which increased during osteogenic differentiation. Immunohistochemical analysis suggested positive Notch2 expression at the ossification front. Down-regulation of Notch2 expression decelerated osteogenic differentiation of ligamentum flavum cells, and Notch2 overexpression promoted osteogenic differentiation of ligamentum flavum cells. Expression of Runx2 and Osterix increased in a manner similar to that of Notch2 during osteogenic differentiation of ligamentum flavum cells, and Notch2 knockdown and overexpression influenced their expression levels. Notch signaling plays an important role in OLF, and Notch may affect the osteogenic differentiation of ligamentum flavum cells via interactions with Runx2 and Osterix.ß

MiR‐199b‐5p inhibits osteogenic differentiation in ligamentum flavum cells by targeting JAG1 and modulating the Notch signalling pathway

“…Moreover, JAG1 knockdown also inhibited Notch signalling, thus suggesting that osteogenic differentiation is executed through the Notch signalling pathway, with JAG1 being one of the principal Notch ligands. Additionally, JAG1 was found to associate with Notch2, but not Notch1, during osteogenic differentiation in human ligamentum flavum cells, which is consistent with our previous study .…”

“…While various signalling pathways have been connected with the regulation of osteogenic differentiation in general, some have also been associated with OSL pathogenesis [6,7,[13][14][15][16]. Of these, the Notch signalling pathway, which influences proliferation, differentiation and mineralization in osteoblasts, has been found to encourage osteogenic differentiation in ligamentum flavum cells [17,18].…”

MiR-132-3p Regulates the Osteogenic Differentiation of Thoracic Ligamentum Flavum Cells by Inhibiting Multiple Osteogenesis-Related Genes

“…Each value was normalized to that of RnU6. Reverse transcription and qPCR for the mRNA levels of FOXO1 , GDF5 and SOX6 were carried out as described previously [49]. Expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and relative gene expression levels were calculated using the 2-ΔΔ C t method.…”

“…Sections for immunohistochemical staining were carried out as described previously [49]. The primary rabbit anti-human antibodies were: anti- FOXO1 (1:200; ab39670, Abcam); anti- GDF5 (1:200; ab93855, Abcam); anti- SOX6 (1:200; ab30455, Abcam).…”

New Horizons in Spine Research: Disc biology, spine biomechanics and pathomechanisms of back pain

“…Bacterial infection in immune‐privileged IVDs can induce inflammation, induce IVD degeneration and result in modic‐like changes in the adjacent marrow in a model system providing some evidence for this provocative and intriguing back pain mechanism. Nerve ingrowth and vascularization of IVDs are also attributed to discogenic pain, and spine pain can be associated with pathology surrounding structures including facet joints and ligamentum flavum …”