n-(n-(3-5-difluorophenacetyl)alanyl)phenylglycine-tert-butyl-ester and Osteoarthritis

During osteoarthritis (OA), chondrocytes undergo de-differentiation, resulting in the acquisition of a fibroblast-like morphology, decreased expression of collagen type II (colII) and aggrecan, and increased expression of collagen type I (colI), metalloproteinase 13 (MMP13) and nitric oxide synthase (eNOS). Notch signaling plays a crucial role during embryogenesis. Several studies showed that Notch is expressed in adulthood.. The aim of our study was to confirm the involvement of Notch signaling in human OA at in vitro and ex vivo levels.. Normal human articular chondrocytes were cultured during four passages either treated or not with a Notch inhibitor: DAPT. Human OA cartilage was cultured with DAPT for five days. Chondrocytes secreted markers and some Notch pathway components were analyzed using Western blotting and qPCR.. Passaging chondrocytes induced a decrease in the cartilage markers: colII and aggrecan. DAPT-treated chondrocytes and OA cartilage showed a significant increase in healthy cartilage markers. De-differentiation markers, colI, MMP13 and eNOS, were significantly reduced in DAPT-treated chondrocytes and OA cartilage. Notch1 expression was proportional to colI, MMP13 and eNOS expression and inversely proportional to colII and aggrecan expression in nontreated cultured chondrocytes. Notch ligand: Jagged1 increased in chondrocytes culture. DAPT treatment resulted in reduced Jagged1 expression. Notch target gene HES1 increased during chondrocyte culture and was reduced when treated with DAPT.. Targeting Notch signaling during OA might lead to the restitution of the typical chondrocyte phenotype and even to chondrocyte redifferentiation during the pathology.

Topics: Basic Helix-Loop-Helix Transcription Factors; Cartilage, Articular; Cell Culture Techniques; Cell Dedifferentiation; Cell Differentiation; Chondrocytes; Collagen Type II; Dipeptides; Homeodomain Proteins; Humans; Osteoarthritis; Protein Transport; Receptors, Notch; Signal Transduction; Transcription Factor HES-1

Here we examined the involvement of Notch signaling in the endochondral ossification process, which is crucial for osteoarthritis (OA) development. Intracellular domains of Notch1 and -2 were translocated into the nucleus of chondrocytes with their differentiation in mouse limb cartilage and in mouse and human OA articular cartilage. A tissue-specific inactivation of the Notch transcriptional effector recombination signal binding protein for Ig kappa J (RBPjκ) in chondroprogenitor cells of SRY-box containing gene 9 (Sox9)-Cre;Rbpj(fl/fl) mouse embryos caused an impaired terminal stage of endochondral ossification in the limb cartilage. The RBPjκ inactivation in adult articular cartilage after normal skeletal growth using type II collagen (Col2a1)-Cre(ERT);Rbpj(fl/fl) mice by tamoxifen injection caused resistance to OA development in the knee joint. Notch intracellular domain with the effector RBPjκ stimulated endochondral ossification through induction of the target gene Hes1 in chondrocytes. Among the Notch ligands, Jagged1 was strongly induced during OA development. Finally, intraarticular injection of N-[N-(3,5-diflurophenylacetate)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT), a small compound Notch inhibitor, to the mouse knee joint prevented OA development. The RBPjκ-dependent Notch signaling in chondrocytes modulates the terminal stage of endochondral ossification and OA development, representing an extracellular therapeutic target of OA.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Calcium-Binding Proteins; Cartilage; Cell Line; Cell Line, Tumor; Chondrocytes; Collagen Type II; Dipeptides; Fluorescent Antibody Technique; HeLa Cells; Homeodomain Proteins; Humans; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Knee Joint; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Osteoarthritis; Osteogenesis; Receptor, Notch1; Receptor, Notch2; Reverse Transcriptase Polymerase Chain Reaction; Serrate-Jagged Proteins; Signal Transduction; SOX9 Transcription Factor; Transcription Factor HES-1